Figure 1.
Global gene expression differences between iPSCs and ESCs are attenuated following excision of a lentiviral reprogramming cassette.
(A) Schematic representation of the different groups of iPSC/ESC lines subjected to microarray analysis. iPSCs carrying a single copy of a “floxed” STEMCCA vector encoding either three (OKS) or four (OKSM) reprogramming factors were treated with Cre-recombinase to generate transgene-free OKS-Cre and OKSM-Cre iPSC clones. Five subclones of the Sox2-GFP/M2rtTA ESC line were isolated, expanded and included as a control. (B) Principal Components Analysis (PCA) performed on the microarray datasets clearly separates ESCs, transgene-carrying iPSCs (OKS and OKSM) and transgene-free iPSCs (OKS-Cre and OKSM-Cre) into three distinct groups, indicative of similar but distinctive gene expression profiles. Notably, PCA is unable to discriminate iPSCs generated with 3 or 4 factors, both before and after transgene removal. Instead, the presence of the transgenes appears to be a major factor influencing the iPSC transcriptome. (C) qRT-PCR measurement of the residual transcriptional activity of the reprogramming vector demonstrates differences in expression across the iPSC lines that correlate with the degree of gene dysregulation revealed by PCA. (D) Hierarchical clustering of the 2,327 genes significantly different between transgene-carrying iPSCs and transgene-free iPSCs (two-way ANOVA, FDR-adjusted p-value <0.01) reveals distinct patterns of gene expression specific to each of the three groups and increased similarity of iPSCs to ESCs following transgene excision.
Figure 2.
Epigenetic status and transcriptional activity of the Gtl2 locus in iPSC lines generated with STEMCCA.
(A) Gtl2 transcript levels were estimated by qRT-PCR in the starting cell population (TTFs), ESCs, and the 20 iPSC clones profiled by microarray. The percentage of methylated CpG dinucleotides in the Gtl2 IG-DMR and Gtl2 DMR were determined by pyrosequencing of sodium bisulfite-treated genomic DNA. Asterisks indicate Gtl2OFF iPSC clones. (B) Twenty additional iPSC clones were derived from TTFs isolated from a different mouse strain (C57BL/6), using either the Doxycycline (Dox)-inducible or the constitutive STEMCCA vector and analyzed as in (A). Doxycycline was withdrawn at the indicated time points or kept throughout the expansion of iPSCs. n.d.: not detected.
Figure 3.
Klf4 is recruited to the Gtl2 promoter region during reprogramming to pluripotency.
(A) Putative binding sites for the Yamanaka factors were identified in the Gtl2 promoter region using ChIP-seq data. (B) ChIP-qPCR analysis confirmed increased binding of Klf4 to the Gtl2 imprinted domain in transgene-carrying iPSCs. Cross-linked protein-DNA complexes were immunoprecipitated from whole cell extracts of ESC/iPSCs using an anti-Klf4 or a control antibody and DNA within the precipitates was isolated and amplified using primers specific for the Gtl2 promoter. Results are shown as fold-enrichment relative to the control antibody.
Figure 4.
Directed differentiation of transgene-free iPSCs into hepatic progenitors is not affected by the epigenetic status of the Dlk1-Dio3 gene cluster.
qRT-PCR analysis of the differentiating cultures at different time points demonstrates induction of the endoderm marker Sox17 followed by an increase in expression of the liver markers AFP and albumin. Remarkably, all iPSC clones displayed an in vitro differentiation capacity comparable to ESCs regardless of the expression levels of Gtl2 and Dlk1.